Issued  June  21,  liK)y. 


.  S.  DEPARTMENT  OF /AGRICULTURE, 

BUREAU  OI?/aNIMAL  INDUSTRY.— Bulletin  H3. 

A.  D.  MELVIN,  Chief  of  Bureau. 


i  =  ILTRATION  EXPERIMENTS  WITH 
~       BACILLUS  CHOLERA  SUIS. 


ASSN. 
^TLAKE  AVE. 
/^lOS  ANI 

BY 


a  N.  McBRYDE,  M.  D., 

Senior  Bacteriohgisiy  Biocheniic  Division. 


%^^\ 


WASHINGTON: 

GOVHRNMENT    PRINTING    OFlilCE. 

1909. 


OFKl 


Issued  June  21,  1909. 

U.  S.  DEPARTMENT  OF  AGRICULTURE, 

BUREAU  OF  ANIMAL  INDUSTRY.— Bulletin  113. 

A.  D.  MELVIN,  Chief  of  Bureau. 


FILTRATION  EXPERIMENTS  WITH 
BACILLUS  CHOLERA  SUIS. 


BY 
C.  N.  McBRYDE,  M.  D., 

Senior  Bacteriologist,  Biochemic  Division. 


LIBRARY  OF  Tht 
LA.CO.  MEDiQi^^oiiM. 
634  SOUTH  WESTLAKE  AVE. 
LOS  ANGELES 


BARLOW  MEDICAL  LIBRARY 
742  NORTH  BROADWAY 
LOS  ANGELES.  CAL 


WASHINGTON : 
GOVERNMENT    PRINTING    OFFICE. 

1909. 


THE  BUREAU  OF  ANIMAL  INDUSTRY. 


Chief:  A.  D.  Melvin. 

Assistant  Chiej:  A.  M.  Farrington. 

ChieJ  Clerk:  Charles  C.  Carroll. 

Biochemic  Division:  M.  Dorset,  chief;  James  A.  Emery,  assistant  chief. 

Dairy  Division:  B.  H.  Rawl,  chief. 

Inspection  Division:  Rice  P.  Steddom,  chief;  Morris  Wooden,  R.  A.  Ramsay,  and 
Albert  E.  Behnke,  associate  chiefs. 

Pathological  Division:  John  R.  Mohler,  chief;  Henry  J.  Washburn,  assistant 
chief. 

Quarantine  Division:  Richard  W.  Hickman,  chief. 

Zoological  Division:  B.  H.  Ransom,  chief. 

Experiment  Station:  E.  C.  Schroeder,  superintendent;  W.  E.  Cotton,  assistant. 

Animal  Hvsbandman:  George  M.  Rommel. 

Editor:  James  M.  Pickens. 

BIOCHEMIC  DIVISION. 

Chief:  M.  Dorset. 

Assistant  Chief:  James  A.  Emery. 

Hog  cholera  investigations:  Chief  of  Division  in  charge;  W.  B.  Niles,  veterinary 
inspectbr  in  charge  of  field  experiments. 

Investigations  of  dips  and  disinfectants:  R.  M.  Chapin,  senior  biochemist  in  charge. 

Bacteriological  investigations  of  meat  food  products:  C.  N.  McBryde,  senior  bacteri- 
ologist in  charge. 

Tuberculin  and  mallein  prepatation  and  distribution:  A.  M.  West,  bacteriologist  in 
charge. 

Meat  inspection  laborataries:  Central  laboratory,  Washington,  D.  C,  T.  M.  Price, 
senior  biochemist  in  charge.  Branch  laboratories:  Chicago,  HI.,  Ralph  Hoagland  in 
charge;  New  York,  X.  Y.,  A.  H.  Roop  in  charge;  Kansas  City,  Kans.,  C.  H.  Swanger 
in  charge;  South  Omaha,  Nebr.,  W.  B.  Smith  in  charge;  East  St.  Louis,  111.,  E.  A. 
Boyer  in  charge;  San  Francisco,  Cal.,  A.  E.  Graham  in  charge. 
2 


LETTER  OF  TRANSMITTAL. 


U.  S.  Department  of  Agriculture, 

Bureau  of  Animal  Industry, 
Washington,  D.  C,  January  30,  1909. 
Sir:  I  have  the  honor  to  transmit  herewith,  and  to  recommend  for 
pubHcation  as  a  bulletin  of  this  Bureau,  the  accompanying  manu- 
script entitled  ''Filtration  Experiments  with  Bacillus  cholerse  sms," 
by  Dr.  C.  N.  McBryde,  of  the  Biochemic  Division. 

The  subject-matter  of  the  paper,  while  technical  in  its  character, 
has  a  direct  bearing  on  the  hog  cholera  problem.  In  a  previous 
publication  issued  by  this  Bureau  (Bulletin  72,  The  Etiology  of  Hog 
Cholera)  it  was  first  shown  that  the  so-called  hog  cholera  bacillus, 
B.  cliolerse.  suis,  could  not  be  regarded  as  the  definite  and  sole  cause 
of  hog  cholera,  since  the  disease  could  readily  be  produced  by  an 
ultra-microscopic  virus  in  the  blood  of  sick  hogs,  which  vims  had 
repeatedly  passed  through  a  filter  that  effectually  prevented  the 
passage  of  B.  cholern-  suis. 

This  discovery  naturally  caused  widespread  discussion  among 
scientific  investigators  upon  this  subject  throughout  the  world, 
some  of  whom  have  questioned  the  accuracy  of  the  findings  pub- 
lishe<l  in  the  bulletin  referred  to.  The  experiments  described  in  the 
present  paper  have  been  carried  out  to  meet  these  criticisms  and 
have  resulted  in  confirming  the  correctness  of  the  work  previously 
published. 

Respectfully, 

A.  D.  Melvin, 
Chief  of  Bureau. 
lion.  James  Wilson, 

Secretary  of  Agriculture. 


CONTENTS. 


Page. 

Introductory 5 

Filters  used 7 

Arrangement  of  filters 7 

Technique  employed  in  filtrations 8 

Cultures  used 9 

Experiments  with  Berkefeld  filters 11 

Summary  of  experiments  with  Berkefeld  filters 13 

Discussion  of  Berkefeld  filtrations 13 

Experiments  with  Paateur-Chamberland  filters 14 

Summary  of  experiments  with  Pasteur-Chamberland  filters 25 

Discussion  of  Pasteur-Chamberland  filtrations 26 

Rate  of  filtration  of  Berkefeld  and  Pasteur-Chamberland  filters 28 

Granule  formation  in  cultures  of  B.  cholerse  suis 29 

Summary  of  results 30 

Conclusions 31 


ILLUSTRATION. 


Page. 
Fig.  1.  Apparatus  for  fractional  filtration,   designed   for  use  with   Pasteur- 
Chamberland  or  Berkefeld  filters 8 

4 


FILTRATION  EXPERIMENTS  WITH  BACILLUS  CHOLERJ]  SUIS. 


INTRODTJCTORY. 

In  the  early  part  of  1905  Doi-set,  Bolton,  and  the  writer*  published 
a  paper  on  the  etiology  of  hog  cholera,  in  which  it  was  first  definitely 
shown  that  hog  cholera  is  due  to  a  filterable  virus  and  not  to  Bacillus 
cholerse  suis  as  had  been  previously  supposed .  The  conclusions  reached 
in  that  paper  were  based  on  an  extensive  series  of  filtration  experi- 
ments in  which  the  blood  serum  of  hogs  sick  of  hog  cholera  was  filtered 
through Berkef eld  antlChamberland  filters  in  order  to  excluded,  cholerse 
suis.  In  these  experiments  a  large  number  of  nonimmune,  healthy  hogs 
were  injected  with  the  filtered  serum,  which  was  carefidly  tested  in 
every  instance  prior  to  injection  for  B.  cholerse  suis.  The  filtrates 
were  proven  to  be  free  from  that  organism  by  either  incubating  the 
entire  filtrate  or  else  taking  out  large  portions  for  subcultures,  and 
also  by  the  injection  of  large  portions  into  guinea  pigs  and  rabbits, 
animals  which  are  extremely  susceptible  to  B.  cholerse  suis.  These 
experiments  with  the  filtered  serum  of  hogs  sick  of  hog  cholera  and 
proven  to  be  free  from  B.  cholerse  suis  showed  that  if  such  serum 
was  injected  subcutaneously  into  well  hogs  it  caused  typical  hog 
cholera  with  great  regularity,  whereas  the  same  serum  injected  into 
small  laboratory  animals  which  are  naturally  very  susceptible  to 
B.  cholerse  suis  was  entirely  without  effect.  In  many  of  these  experi- 
ments rabbits  were  injected  with  as  much  as  20  c.  c.  of  the  filtrates 
without  any  subsequent  ill  effects,  whereas  hogs  injected  with  like 
amounts  of  the  filtrates  died  of  typical  hog  cholera. 

These  results,  coupled  with  others  which  developed  in  the  course 
of  this  investigation,  led  to  the  following  conclusions:  (1)  That  in  the 
disease  known  as  hog  cholera  there  is  some  other  etiological  factor 
present  besides  B.  cholerse  suis;  (2)  that  this  other  factor  is  an  ultra- 
visible  virus  sufficiently  small  to  pass  through  the  pores  of  Berkefeld 
and  Chamberland  filters;  {'])  that  this  ulti-a-visil)le  virus  is  tiie  true 
cause  of  hog  cliolera,  and  that  B.  choleras  suis,  when  present  in  the 
blood  and  tissues  of  animals  sick  of  hog  cholera,  is  pro])ably  a  sec- 
ondary invader  and  at  most  an  accessory  factor  in  the  (Hsease. 

These  ccmclusions  with  regard  to  the  etiology  of  hog  cholera,  being 
decidedly  revolutionary  in  character,  attracted  wides])read  attention 

"Dorset,  M.,  Bolton,  B.  M.,  and  McBryde,  C.  N.  The  Etiology  <jf  Ilog  Cholera. 
U.  S.  Department  of  Agriculture,  Bureau  of  Animal  Industry,  Bulletin  72.  Wash- 
ington.  1905. 

6 


6  FILTRATION   EXPEBIMENTS   WITH   B.   CHOLERffil  SUIS.      * 

among  scientific  investigators  in  other  countries,  and  it  is  gratifying 
to  state  that  our  results  have  been  confirmed  by  many  well-known 
workers  in  various  parts  of  the  world.  Thus  in  Germany  the  same 
results  were  obtained  by  Ostertag  and  Stadie  in  the  Hygienic  Institute 
of  the  Veterinary  High  School  at  Berlin,  and  by  Uhlenhuth  and  his 
coworkers,  Hiibener,  Xylander,  and  Botz,  in  the  laboratories  of  the 
Imperial  Board  of  Health  at  Berlin.  Similar  results  were  obtained 
by  Hutyra  in  Austria,  by  Stockman  and  McFadyean  in  England,  by 
Theiler  in  South  Africa,  and  within  the  past  year  b}'- Carre,  Leclainche, 
and  Valine  in  France.  In  our  own  country  our  results  have  been 
confirmed  by  McClintock,  Boxmeyer,  and  Siffer.  It  is  thus  seen 
that  our  conclusions  with  regard  to  the  etiology  of  hog  cholera  are  not 
lacking  in  confirmatory  proof. 

Certain  criticisms,  however,  have  been  made  of  our  earlier  work  on 
the  etiology  of  hog  cholera,  referred  to  above.  In  1907  Lourens," 
subdirector  of  the  State  Serum  Institute  at  Rotterdam,  published 
an  article  on  the  fiJterability  of  Bacillus  suipestifer  in  which  he  makes 
the  statement  that  the  so-called  hog-cholera  bacillus,  known  as  B. 
cTiolerse  suis  or  B.  suipestifer,  is  capable  of  passing  through  filters  of 
the  Chamberland  and  Berkefeld  types,  composed,  respectively,  of 
unglazed  porcelain  and  infusorial  earth.  He  claims  that  the  ability 
of  B.  cholerse,  suis  to  pass  through  filters  of  the  types  mentioned  is 
due  to  a  property  which  this  bacillus  possesses  of  breaking  up  into 
granules  sufficiently  small  to  pass  through  the  pores  of  the  filter. 
He  also  asserts  that  B.  cholerse  suis  is  the  cause  of  hog  cholera  and 
that  none  of  the  investigators  who  have  conducted  filtration  experi- 
ments to  show  that  hog  cholera  is  due  to  a  filterable  virus  have 
afforded  sufficient  and  convincing  proof  that  the  filtrates  which  they 
employed  did  not  contain  B.  cholerse  suis. 

While  we  had  no  doubts  as  to  the  accuracy  of  our  earlier  filtration 
work,  nor  any  doubt  whatever  that  hog  cholera  is  due  to  a  filterable 
virus,  especially  since  our  earlier  work  has  been  confirmed  by  such 
well-known  investigators  as  those  cited  above,  we  nevertheless  felt 
that  such  criticisms  should  not  be  allowed  to  pass  unnoticed,  and  it 
was  vnth.  a  view  to  meeting  these  criticisms  that  the  experiments 
described  in  the  present  paper  were  undertaken. 

In  connection  with  our  earlier  work  on  the  etiology  of  hog  cholera  the 
writer  carried  out  a  series  of  filtration  experiments  with  cultures  of 
B.  cholerse  suis.  This  was  done  as  a  further  test  of  the  efficiency  of 
the  filters  used — that  is,  to  determine  whether  filters  of  the  Berkefeld 
and  Chamberland  types  could  be  relied  upon  absolutely  to  restrain 
or  keep  back  B.  cholerse  suis. 

a  Lourens,  Louis  F.  D.  E.  Untersuchungen  iiber  die  Filtrierbarkeit  der  Schweine- 
pest-bacillen  (Bac.  suipestifer).  Centralblatt  fiir  Bakteriolc^ie,  abt.  1,  orig.,  band  44, 
heft  5,  pp.  420-427,  Aug.  20;  heft  6,  pp.  504-512,  Aug.  31;  heft  7,  pp.  630-648,  Sept.  17. 
Jena,  1907, 


DESCRIPTION    AND    ARRANGEMENT    OF    FILTERS.  7 

Since  the  publication  of  Lourens's  paper  a  second  series  of  filtra- 
tion experiments  with  bouillon  cultures  of  B.  cholerse  suis  has  been 
carried  out  by  the  writer,  and  the  results  obtained,  together  with 
those  obtained  in  the  earlier  experiments  referred  to  above,  none  of 
which  have  hitherto  been  published,  are  now  recorded  in  detail. 

FILTERS  USED. 

The  filters  used  were  Berkefeld  laboratory  cylinders  Nos.  5  and  7, 
and  Pasteur-Chamberland  bougies  ''F"  and  "B,"  and  were  of  the 
same  types  as  those  used  in  the  filtration  experiments  described  in 
Bulletin  72. 

A  circular  which  accompanied  the  Pasteur-Chamberland  bougies 
stated  that  these  filters  were  manufactured  in  France  and  imported 
into  this  country,  and  this  was  also  stated  on  the  cardboard  boxes  in 
which  the  bougies  were  packed.  The  individual  bougies  were  labeled 
in  French  as  follows:  "F  [or  "B"l  Filtre  Chamberland,  Systeme 
Pasteur,  H.  B.  &  Cie.,  Choisy-le-Roi." 

New  filters  were  used  for  each  experiment,  except  in  the  case  of 
experiments  7  and  8  with  the  Pasteur-Chamberland  filters,  whete 
filters  were  employed  which  had  been  used  once  and  then  cleansed 
according  to  the  methods  adopted  by  Lourens. 

Before  being  used  the  filters  were  first  tested  by  submerging  them 
in  water  and  then  forcing  compressed  air  through  their  walls.  If 
the  air  came  through  the  walls  of  the  filter  in  the  form  of  small  bubbles 
or  beads  the  filter  was  regarded  as  being  free  from  flaws,  but  if  large 
air  bubbles  formed  on  the  sides  this  was  taken  as  an  indication  of 
possible  defects  or  flaws,  and  such  filters  were  discarded.  This 
method  of  testing  the  filters  is  similar  to  that  employed  by  Lourens. 

ARRANGEMENT    OF    FILTERS. 

In  the  first  three  experiments  with  Berkefeld  filters  and  the  first 
three  experiments  with  the  Pasteur-Chamberland  ty])e  the  filters 
were  attached  to  ordinary  side-arm  or  suction  flasks  and  the  filtrates 
collected  in  one  portion.  In  the  remainder  of  the  experiments  the 
fractional  method  of  filtration  was  used,  for  wliich  a  special  form  of 
apparatus  was  devised.  The  apparatus,  which  is  shown  in  figure  1, 
may  be  used  with  either  the  Berkefeld  or  the  Chamberland  ty])e  of 
filter.  It  coiLsists  essentially  of  a  double  side-arm  suction  flask  with 
which  the  filters  are  connected  above  by  means  of  rubber  stoppers 
and  a  protected  outlet  below  connected  with  the  lower  end  of  the 
suction  flask  by  a  short  [)iece  of  heavy  black  rubber  tubing.  The 
outlet  consists  of  a  glass  tube  surrounded  by  a  glass  sliield  which  Ls 
open  at  the  lower  end  and  is  plugged  with  cotton  to  protect  the  out- 
let tube  from  dust.     The  outlet  is  controlled  by  a  pinchcock  above. 


8 


FILTRATION   EXPERIMENTS   WITH   B.    CHOLERiE   SUIS. 


One  arm  of  the  suction  iiask  is  connected  by  means  of  heavy  rubber 
tubing  with  a  vacuum  gauge  and  suction  pump.  The  other  arm  is 
connected  by  means  of  rubber  tubing  with  a  glass  inlet  tube  plugged 
with  cotton,  the  admission  of  air  into  the  suction  flask  being  con- 
trolled by  means  of  a  screw  pinchcock.     (See  fig.  1.) 


Fig.  1.— Apparatus  for  fractional  filtration,  designed  for  use  with  Pasteur-Chamberlaiid  or  Berkefeld 
filters,  a,  Glass  mantle  surrounding  filter;  6,  Chamlierland  filter;  c,  paraffin  joint;  d  and  e,  rubber 
stoppers;/,  double  side-arm  suction  flask;  g,  pinchcock  controlling  outlet  from  suction  flask;  ft,  outlet 
tube  surrounded  by  glass  shield  and  attached  to  lower  end  of  suction  flask  by  means  of  short  rubber 
tubing;  i,  glass  shield  fused  to  and  surrounding  outlet  tul)e  as  a  protection  against  contamination  when 
the  filtrates  are  drawn  off;  j,  glass  inlet  tuiie  plugged  with  cotton,  for  admitting  air  into  suction  flask;  k, 
pinchcock  governing  the  admission  of  air  into  flask;  /,  vacuum  gauge;  m,  stopcock  connected  with 
vacuiun  pump. 

TECHNIQUE    EMPLOYED    IN    FILTBATIONS. 

In  conducting  a  filtration  with  the  apparatus  described,  the  pinch- 
cocks  g  and  fc  are  first  closed  off  and  the  vacuum  applied  at  m.  The 
filtration  is  then  allowed  to  proceed  until  the  filtrate  reaches   the 


TECHNIQUE   EMPLOYED    AND    CULTURES    USED.  9 

desired  level  in  the  suction  flask  /.  As  soon  as  the  suction  flask  is 
exhausted  of  air  the  rubber  tubing  above  the  pinchcock  g  will  be 
seen  to  collapse  and  will  remain  so  as  long  as  the  vacuum  is  applied. 
When  the  filtrate  reaches  the  desired  level  in  the  suction  flask  the 
vacuum  is  closed  off  and  the  air  gradually  admitted  into  the  suction 
flask  by  means  of  the  pinchcock  Ar  through  the  glass  inlet  tube  plugged 
with  cotton  {j) .  The  admission  of  air  into  the  suction  flask  may  be  reg- 
ulated by  watching  the  rubber  tubing  above  the  pinchcock  g,  which 
expands  as  soon  as  the  vacuum  within  the  suction  flask  has  been 
neutralized.  The  filtrate  which  has  collected  in  the  suction  flask  is 
now  drawn  off  into  a  sterile  Erlenmeyer  flask  by  means  of  the  pinch- 
cock g  through  the  glass  outlet  tube  Ji.  .  In  withdrawing  the  filtrate 
an  assistant  removes  the  cotton  plug  from  the  glass  shield  surround- 
ing the  outlet  tube,  steadying  the  shield  at  the  same  time.  The  neck 
of  the  Erlenmeyer  flask,  which  is  to  receive  the  filtrate,  is  then 
introduced  within  the  shield  beneath  the  outlet  tube  and  the  filtrate 
allowed  to  flow  out  by  releasing  the  pinchcock  above.  The  inlet,  tube 
is  now  closed  off  again  by  means  of  the  pinchcock  k,  the  vacuum 
reapplied  at  m,  and  another  portion  of  the  filtrate  collected  and  drawn 
off  as  before.  In  this  way  as  many  fractions  may  be  collected  as  are 
desired. 

Dry  heat  is  used  for  the  sterilization  of  all  glass  portions  of  the 
apparatus  except  the  glass  mantle  a  surrounding  the  filter,  which 
being  outside  of  the  filter  does  not  require  sterilization.  Both  arms 
of  the  suction  flask  should  be  plugged  with  cotton  to  prevent  dust 
particles  from  being  drawn  into  the  flask.  The  filters  and  all  rubber 
connections  are  sterilized  by  boiling  in  distilled  water  for  from  one- 
half  hour  to  one  hour.  After  the  apparatus  is  set  up  all  joints  should 
be  carefully  paraffined. 

CULTURES  USED. 

Five  different  strains  of  Bacillus  cholerse  suis  were  used.  These 
cultures  were  obtained  from  different  sources,  either  directly  from 
hogs  which  died  of  hog  cholera  or  from  guinea  pigs  inoculated  with 
cultures  obtained  from  hogs  which  had  died  of  hog  cholera.  The 
cultures  were  grown  on  the  various  laboratory  media  and  their 
biological  and  mor])hological  characters  carefully  noted.  All  of  the 
cultures  used  were  virulent  for  guinea  pigs  and  rabbits. 

A  brief  record  of  the  different  cultures  is  appended. 

1.  Culture  Gp  3998  S. — This  culture  was  obtained  from  guinea  pig 
3998,  which  tlied  from  the  inoculation  of  a  culture  obtained  from  hog 
1086.  Ilog  1086  was  a  nonimmune  animal  which  served  as  a  check 
in  the  Iowa  experiments.  The  animal  was  e.xposetl,  together  with 
certain  other  treated  hogs,  to  a  natural  outbreak  of  hog  cholera.  As 
a  result  of  the  exi)()sure  the  animal  c<jntracted  the  disease  and  (li(>d 
with  typical  cholera  lesions,  B.  cholerx  suis  being  obtained  in  pure 
72369— Bull.  113-09 2 


10  FILTRATION   EXPERIMENTS   WITH  B.   CHOLERiE  SUIS. 

culture  from  the  heart  blood  and  spleen.  A  subculture  obtained 
from  agar  plates  matle  from  the  spleen  of  hog  1086  was  used  for  the 
inoculation  of  guinea  pig  3998.  The  guinea  pig  died  within  six  days, 
and  B.  cholerse  suis  was  recovered  in  pure  culture  from  the  spleen  and 
heart  blood.  A  subculture  from  agar  plates  made  from  the  spleen  of 
guinea  pig  3998  was  cultivated  on  the  various  laboratory  media  and 
found  to  correspond  with  B.  cholerse  suis  in  all  respects.  This  culture, 
which  we  have  designated  Gp  3998  S,  was  used  in  the  first  four 
experiments  with  Berkefeld  filters,  and  the  first  two  experiments 
with  Pasteur-Chamberland  filters. 

2.  Culture  Gp469£  S. — Obtained  from  the  spleen  of  guinea  pig  4692, 
which  died  from  the  injection  of  1  c.  c.  of  unfiltered,  undiluted  blood 
seioim  of  hog  1208,  an  experiment  hog  carrying  the  Scribner  strain 
of  disease  in  the  experiments  at  Washington  (see  "Experiments  with 
tail  blood,"  Bulletin  72,  page  38).  This  culture  was  used  in  a  num- 
ber of  the  culture  experiments  described  in  Bulletin  72  and  was 
found  to  correspond  with  5.  cholerse  suis  in  all  of  its  cultural  characters. 

3.  Culture  H  2199  S. — This  culture  was  obtained  directly  from  hog 
2199,  which  served  as  a  check  in  the  Iowa  immunity  experiments. 
The  animal  died  from  the  injection  of  2  c.  c.  of  unfiltered  disease- 
producing  blood  from  the  Scribner  and  Syphax  strains  of  disease.  A 
description  of  the  Scribner  and  Syphax  outbreaks  may  be  found  in 
Bulletin  102"  of  this  Bureau.  The  autopsy  on  hog  2199  revealed 
typical  cholera  lesions  and  B.  cholerse.  suis  was  obtained  in  pure  culture 
from  the  spleen.  This  culture,  designated  as  H  2199  S,was  cultivated 
on  the  different  laboratory  media  and  was  found  to  correspond  in  all 
of  its  cultural  characters  with  B.  cholerse  suis. 

4.  Culture  II  2^50  S. — This  was  obtained  directl}^  from  hog  2450,  a 
western  hog  used  in  the  Iowa  experiments.  Hog  2450  was  an  unin- 
oculated  check  and  was  placed  in  the  exposure  pen  (for  description  of 
exposure  pen  see  Bulletin  102,  page  12)  along  with  certain  treated 
hogs  in  order  to  test  the  virulence  of  the  disease  prevailing  in  the  expo- 
sure pen  at  the  time.  The  animal  contracted  hog  cholera  in  the  usual 
time  and  died  with  characteristic  lesions  of  the  disease.  Cultures 
from  the  spleen  were  grown  on  the  cjifferent  laboratory  media  and 
found  to  correspond  in  all  respects  with  B.  cholerse  suis. 

5.  Culture  II  2228  S. — This  culture  was  obtained  directly  from 
hog  2228,  which  died  as  a  result  of  an  injection  of  5  c.  c.  of  defibrinated 
blood  obtained  from  an  outbreak  of  hog  cholera  in  Iowa.  Hog  2228 
exhibited  characteristic  cholera  lesions  at  autopsy  and  agar  plates 
from  the  spleen  revealed  B.  cholerse  suis.  This  culture,  like  all  of  the 
others,  was  cultivated  on  the  various  laboratory  media  and  was 
found  to  correspond  with  B.  cholerse  suis  in  all  of  its  cultural  characters. 

a  Dorset,  M.,  McBryde,  C.  N.,  and  Niles,  W.  B.  Further  Experiments  Concerning 
the  Production  of  Immunity  from  Hog  Cholera.  U.  S.  Department  of  Agriculture, 
Bureau  of  Animal  Industry,  Bulletin  102.    Washington,  1907. 


BERKEFELD   FILTER   EXPERIMENTS.  11 

EXPERIMENTS  WITH  BERKEFELD  FILTERS. 

EXPERIMENT  1. 

A  flask  containing  400  c.  c.  of  neutral  beef  broth  "was  inoculated  with 
culture  Gp  3998  S  and  placed  in  the  incubator.  At  the  end  of  twenty- 
four  hours  the  flask  was  well  clouded  and  200  c.  c.  of  the  culture  was 
then  passed  through  a  Berkefeld  laboratory  cylinder  No.  5.  The 
vacuum  used  was  20  inches  and  the  time  occupied  in  filtration  was 
one  and  one-half  hours.  The  filtrate  was  collected  in  one  portion  in 
an  ordinary  side-arm  flask  and  placed  at  once  in  the  incubator.  At 
the  end  of  eighteen  hours  the  filtrate,  which  came  through  the  filter 
perfectly  clear,  was  distinctly  clouded.  Agar  plates  from  the  filtrate 
showed  B.  cholerse  suis  in  pure  culture.  A  subculture  made  from 
the  agar  plates  was  grown  on  the  different  laboratory  media  and 
found  to  correspond  in  all  cultural  characteristics  with  the  original 
culture.  The  results  obtained  in  this  experiment  show  that  B. 
cholerse  suis  passed  through  the  Berkefeld  filter  in  the  first  200  c.  c. 
of  filtrate. 

EXPERIMENT  2. 

A  flask  of  neutral  beef  broth  was  inoculated  with  culture  Gp  3998  S 
and  incubated  at  37°  C.  for  twenty-four  hours.  The  culture  showed 
well-marked  clouding  and  a  microscopic  examination  showed  the 
organisms  to  be  actively  motile.  Filtration  was  made  through  a 
Berkefeld  laboratory  cylinder  No.  7.  The  amount  of  culture  filtered 
was  100  c.  c.  and  the  time  occupied  in  filtration  was  one  and  one-half 
hours.  The  filtration  was  conducted  under  a  vacuum  of  22  inches. 
The  filtrate  was  collected  in  one  portion  and  was  immediately  ])laced 
in  the  incubator.  After  twenty-four  hours  in  the  incubator  the 
filtrate  showed  distinct  clouding  and  agar  plates  revealed  a  pure 
culture  of  B.  cholerse  suis.  A  subculture  made  from  the  agar  })lates 
was  cultivated  on  the  different  laboratory  media.  This  culture  was 
found  to  correspond  with  the  original  culture  in  all  of  its  cultural 
characteristics.  In  this  experiment  B.  cholerse  suis  passed  through 
the  Berkefeld  filter  in  the  first  100  c.  c.  of  filtrate. 

EXPERIMENT  3. 

This  experiment  was  ])ractically  a  repetition  of  experiment  2  and 
the  results  obtained  were  the  same.  The  filter  used  was  a  Berkefeld 
laboratory  cylinder  No.  7.  The  amount  of  culture  filtered  was  100 
c.  c,  which  required  one  and  one-half  hours  to  ])ass  through  the  filter. 
The  vacuum  employed  was  23A  inches?.  The  entire  filtrate  was 
collected  in  one  portion,  as  in  the  two  ])rece<ling  experiments.  After 
forty-eight  hours  in  the  incubator  the  filtrate  showed  heavy  clouding 
and  agar  ])lates  revealed  a  ])ure  culture  of  B.  cholene  suis.  A  sub- 
culture was  made  froiu  tlie  agar  j)lates  and  grown  on  the  different 
laboratory  media.  This  culture  was  found  to  correspond  in  all 
cultural  characteristics  with  the  original  culture. 


12 


FILTRATION   EXPERIMENTS   WITH   B.   CHOLER.E  SUIS. 


EXPERIMENT  4. 

In  this  experiment  the  fractional  method  of  filtration  was  used, 
the  filtrates  being  collected  in  large  sterile  test  tubes.  In  carrying 
out  the  experiment  a  twenty-four  hour  culture  of  Gp  3998  S,  grown 
on  neutral  beef  broth,  was  used.  Filtration  was  made  through  a 
Berkefeld  laboratory  cylinder  No.  7.  The  amount  of  culture  filtered 
was  120  c.  c.  and  the  actual  time  consumed  in  the  filtration  was  one 
hour  and  eighteen  minutes.  The  suction  varied  from  20^  to  21^ 
inches.  The  details  of  the  experiment  are  shown  in  the  following 
table : 

Table  showing  details  of  experiment  4- 


Portion. 

Amount. 

Time 
consumed 
in  passing 

filter. 

Suction. 

Result  of  incuba- 
tion. 

I : 

c.  c. 
20 
20 
20 
20 
10 
10 
10 
10 

Minutes. 
4 
7 

10 
12 
7 
10 
11 
17 

Inches. 
20i 
201 
20J 
21 
21 
21 
21J 
21i 

II 

Do. 

Ill 

Do. 

IV 

Do. 

V 

Do. 

VI 

Do. 

VII 

VIII 

Do. 

The  test  tubes  containing  the  filtrates  were  placed  in  the  incubator 
immediately  after  filtration.  At  the  end  of  twenty-four  hours  por- 
tions VII  and  VIII  showed  very  slight  clouding;  other  tubes  remained 
clear.  At  the  end  of  forty-eight  Ijours  portions  VII  and  VIII  showed 
increased  clouding,  while  the  others  remained  clear.  Agar  plates 
were  made  from  portions  VII  and  VIII  and  revealed  pure  cultures  of 
B.  cholerx  suis.  Subcultures  from  the  agar  plates  were  grown  on 
the  various  laboratory  media  and  found  to  correspond  with  the  origi- 
nal culture.  Portions  I  to  VI  were  kept  in  the  incubator  for  two 
weeks  and  remained  j)erfectly  clear.  Hanging-drop  preparations  and 
agar  plates  from  these  })ortions  failed  to  reveal  any  organisms. 

In  this  experiment  the  Berkefeld  cylinder  held  back  the  organisms 
until  100  c.  c.  of  the  culture  had  been  filtered,  but  the  first  portion 
of  10  c.  c.  collected  after  100  c.  c.  of  culture  had  passed  the  filter 
showed  B.  cholerse  suis. 

EXPERIMENT  5. 

In  this  experiment  a  twenty-six  hour  bouillon  culture  (H  2450  S) 
was  used.  The  reaction  of  the  beef  broth  in  this  instance  was  1 
per  cent  acid.  The  culture  showed  well-marked,  uniform  clouding, 
and  a  microscopic  examination  previous  to  filtration  revealed  active 
motility.  Filtration  was  made  through  a  Berkefeld  cylinder  No.  5 
arranged  for  fractional  filtration.  The  vacuum  employed  varied 
from  20  to  23  inches.  It  was  the  intention  to  filter  several  hundred 
cubic  centimeters  of  the  culture  and  collect  the  filtrate  in  separate 


SUMMABY  AND  DISCUSSION   OF   BERKEFELD   FILTRATIONS. 


13 


fractions,  as  in  the  preceding  experiment,  but  the  filtration  proceeded 
verj'^  slowly  and  was  discontinued  at  the  end  of  forty-five  minutes 
after  50  c.  c.  of  culture  had  passed  the  filter.  The  filtrate  was  drawn 
off  in  a  small  sterile  Erlenmeyer  flask  and  placed  in  the  incubator. 
At  the  end  of  eighteen  hours  the  filtrate  showed  a  distinct,  uniform 
clouding  and  agar  plates  revealed  a  pure  culture  of  B.  cholerse.  suis. 
In  this  experiment  B.  cholerse  suis  passed  through  a  Berkefeld 
cylinder  in  the  first  50  c.  c.  of  filtrate. 


SUMMARY    OF    EXPERIMENTS    WITH    BERKEFEI.D    FILTERS. 

The  results  of  the  experiments  with  Berkefeld  filters  are  summarized 
in  the  following  table: 

Summary  of  Berkefeld  filtrations. 


k 

Date 
begun. 

Grade 

of 
filter. 

Culture  used. 

3 

1? 

r 

o  S  H 

©■as 
p.2g 

Vacu- 
um. 

No. 

Age. 

Medium. 

Result. 

1 

2 

1904. 
Feb.  28 

Mar.     7 
Mar.     9 
Mar.   15 

1908. 
June  12 

No.  5- 

No.  7. . 

Gp3998  S... 
do 

Hrs. 
24 

24 
24 

24 

2(5 

Neutral  beet 

broth. 
do 

c.  c. 
200 

100 
100 
120 

50 

ft.    OT. 

1    30 

1    30 
1    30 

1     18 

45 

Inches. 
20 

22 

23i 

20J-21i 

20  -23 

B.   cholerx  suis   in 
filtrate. 
Do. 

3 

4 

No.7..! do 

No.  7.. do 

do 

do 

Do. 

5 

No.  5.! 

1124508 

+  1  beef  broth.. 

filtrate  after  100 
V.  V.  had  passed 
filtor. 
B.   cholerx  suis  in 
fillratu. 

DISCUSSION    OF    BERKEFELD    FILTRATIONS. 

In  the  foregoing  experiments  five  Berkefeld  cylinders,  all  of  them 
new,  were  tested  with  bouillon  cultures  of  B.  cholerse  suis,  and  in  each 
instance  the  organism  passed  through  the  walls  of  the  filter.  In  the 
case  of  one  cylinder  (experiment  4)  the  organism  did  not  appear  in 
the  filter  until  100  c.  c.  of  the  culture  had  been  filtered.  In  one 
instance  (experiment  5)  the  organism  appeared  in  the  first  50  c.  c. 
of  filtrate.  With  two  of  the  cylinders  (experiments  2  and  3)  the 
organism  passed  through  the  walls  of  the  filter  in  the  first  100  c.  c. 
of  filtrate,  and  in  the  case  of  the  remaining  filter  (experiment  1)  the 
organism  appeared  in  the  first  200  c.  c.  of  filtrate. 

These  results  would  indicate  that  in  the  ca.se  of  liquid  cultures  or 
relatively  heavy  suspensions  of  small  micro-organisms  like  B.  cholerse 
suis  the  small  laboratory  Berkefeld  filter  can  not  be  relied  upon  to 
ke«^j)  back  the  organisms  when  any  considerable  amount  of  the 
material  is  filtered. 

That  the  results  obtained  in  these  experiments  conform  with  those 
obtained  by  other  investigators  who  have  testinl  the  Berkefeld  filter 
is  shown  by  the  following  brief  review  of  the  literature  relating  to 
Berkefeld  filters. 


14  FILTBATION   EXPERIMENTS   WITH   B.   CHOLERA  SUIS. 

In  1902  Wherry"  found  that  the  bacillus  of  pneumonia  in  guinea 
pigs,  an  organism  somewhat  shorter  than,  but  very  nearly  as  thick 
as,  B.  clwlerae  mis,  would  pass  through  the  pores  of  a  Berkefeld 
cylinder  No.  5.  Bouillon  cultures  one  and  three  days  old  were  used 
in  these  experiments,  and  the  organism  appeared  in  the  filtrate  after 
from  75  c.  c.  to  80  c.  c.  of  the  culture  had  been  fdtered. 

Pfuhl''  in  1903  made  a  series  of  tests  with  four  small  Berkefeld 
cylinders  and  found  that  of  these  only  one  could  be  depended  on 
with  safety  to  deliver  the  first  100  c.  c.  of  fdtrate  germ  free.  One  of 
the  filters  gave  50  c.  c.  of  germ-free  filtrate,  but  no  more;  the  other 
two  filters  failed  to  give  even  50  c.  c.  of  germ-free  filtrate.  The  cul- 
tures used  in  these  tests  were  B.  coli  communis  and  a  vibrio  of  the 
same  size  as  the  cholera  spirillum. 

In  a  parallel  series  of  experiments  with  large  Berkefeld  cylinders, 
which  have  thicker  walls,  Pfuhl  found  that  50  per  cent  of  these  filters 
were  unable  to  restrain  organisms  approximating  the  size  of  B. 
typhosus  and  B.  dysenteric. 

Novy  antl  MacNeal'^  in  1904  found  that  Trypanosoma  leivisi  could 
be  passed  through  a  Berkefeld  filter. 

Novy  and  Knapp'^  in  1906  showed  that  even  so  large  an  organism 
as  Spirochseta  ohermeieri,  which  is  7  to  19  microns  or  more  in  length, 
will  pass  through  the  small  Berkefeld  filters  under  a  pressure  of  50 
pounds. 

Bulloch,  Craw,  and  Atkin^  in  1908  tested  ten  Berkefeld  filters  with 
tap  water  and  found  that  only  one  gave  a  sterile  filtrate  on  the  first 
day  under  a  maximum  pressure  of  32.5  pounds  to  the  square  inch. 
The  remaining  nine  yielded  contaminated  filtrates  within  fifteen 
minutes,  or  practically  as  soon  as  the  filters  were  started. 

EXPERIMENTS  WITH  PASTETJR-CHAMBERIiAND  FILTERS. 

EXPERIMENT  1. 

A  flask  of  neutral  beef  broth  was  inoculated  with  culture  Gp 
3998  S  and  incubated  for  twenty-four  hours.  At  the  end  of  this 
time  the  culture  showed  well-marked  clouding  and  a  microscopic 

a  WTierry,  William  B.  Experiments  on  the  permeability  of  the  Berkefeld  filter  and 
the  Paateur-Chamberland  bougie  to  bacteria  of  small  size.  Journal  of  Medical  Re- 
search, vol.  8  (n.  s.,  vol.  3),  No.  2,  pp.  322-328.     Boston,  Nov.,  1902. 

b  Pfuhl,  E.  Ergebnisse  einer  erneuten  priifung  einiger  kieselgur-  und  porzellan- 
filter  auf  keimdichtigkeit.  Festchrift  zum  60th  Geburtstag  von  Robert  Koch,  pp. 
75-86.     Jena,  1903. 

cNovy,  F.  G.,  and  MacNeal,  Ward  J.  On  the  filtration  of  trypanosomes.  Michi- 
gan Academy  of  Science,  Sixth  Report,  p.  180.     Lansing,  1904. 

'^Novy,  F.  G.,  and  Knapp,  R.  E.  Studies  on  Spirillum  ohermeieri,  and  related 
organisms.  Journal  of  Infectious  Diseases,  vol.  3,  No.  3,  pp.  291-393.  Chicago, 
May  18,  1906. 

«  Bulloch,  William,  Craw,  J.  Anderson,  and  Atkin,  E.  E.  On  the  relative  efficacy 
of  the  Doulton,  Berkefeld,  and  Brownlow  filters.  Journal  of  Hygiene,  vol.  8,  No.  1, 
pp.  63-69.     Cambridge,  Jan.,  1908. 


EXPERIMENTS   WITH   PASTEUB-CHAMBERLAND   FILTEES.  15 

examination  revealed  active  motility.  The  culture  was  then  passed 
through  an  F  bougie  arranged  to  deliver  into  an  ordinary  side-arm 
suction  flask.  The  amount  of  culture  filtered  was  200  c.  c,  the 
vacuum  employed  was  23  inches,  and  the  time  consumed  in  passing 
through  the  filter  was  thirty-five  minutes.  The  entire  filtrate  was  col- 
lected in  one  portion.  When  the  filtration  was  complete  the  side-arm 
flask  containing  the  filtrate  was  disconnected  from  the  filter,  plugged 
with  a  sterile  cotton  plug,  and  immediately  placed  in  the  incubator. 
At  the  end  of  eight  days  the  filtrate  was  found  to  be  perfectly  clear, 
and  a  careful  microscopic  examination  at  this  time  failed  to  reveal 
any  inicro-organisms.  A  small  portion  of  the  filtrate  was  removed 
at  this  time  by  means  of  a  sterile  pipette  and  the  remainder  replaced 
in  the  incubator.  A  guinea  pig  was  injected  subcutaneously  with 
0.5  c.  c.  of  the  portion  removed  and  showed  no  ill  effects  from  the 
inoculation.  The  filtrate  was  kept  in  the  incubator  for  six  weeks 
and  remained  perfectly  clear.  The  filtrate  was  again  subjected  to  a 
carefid  microscopic  examination  before  being  discarded,  and  was 
apparently  perfectly  sterile. 

EXPERIMENT  2. 

A  flask  containing  400  c.  c.  of  neutral  beef  broth  was  inoculated 
with  culture  Gp  3998  S  and  placed  in  the  incubator  for  twenty-four 
hours.  One-half  of  the  culture  (200  c.  c.)  was  then  passed  through 
an  F  bougie  and  the  other  half  through  a  B  bougie,  the  two  bougies 
being  fitted  to  ordinary  side-arm  suction  flasks.  A  vacuum  of  21 
to  22  inches  was  used,  and  the  time  required  for  the  two  filtrations, 
which  were  conducted  simultaneously,  was  approximately  forty-five 
minutes.  The  two  side-arm  flasks  containing  the  filtrates  were 
immediately  placed  in  the  incubator,  where  they  remained  for  six 
weeks  without  showing  any  signs  of  bacterial  growth.  Before  being 
finally  discarded  both  filtrates  were  subjected  to  a  careful  micro- 
scopic examination,  but  no  micro-organisms  could  be  demonstrated 
in  either  hanging-drop  or  stained  preparations. 

EXPERIMENT  3. 

A  seven-day  bouillon  culture  (Gp  4692  S)  was  used  in  this  experi- 
ment. Filtration  was  made  through  a  Pasteur-Chamberland  B 
bougie.  The  filtration  was  discontinued  after  100  c.  c.  of  filtrate  had 
passed  the  filter.  The  vacuum  gauge  recorded  20  inches  during  the 
filtration,  which  occupied  thirty-seven  minutes.  The  filtrate  was 
collected  in  one  portion  and  immediately  placed  in  the  incubator. 
After  two  weeks  in  the  incubator  the  filtrate  showed  a  very  slight 
opalcvscence,  but  a  careful  microscopic  examination  failed  to  reveal 
any  micro-organisms  and  the  opalescence  was  attributed  to  a  slight 
precipitate  from  the  beef  broth.     In  order  to  test  further  the  ster- 


16 


FILTRATION   EXPERIMENTS   WITH   B.   CHOLERA  SUIS. 


ility  of  the  filtrate,  rabbits  were  injected  as  follows:  Rabbit  1571 
received  1  c.  c.  of  filtrate  intravenously;  rabbit  1332,  3  c.  c.  intra- 
venously; rabbit  1570,  5  c.  c.  intravenously;  rabbit  1574,  5  c.  c. 
intraperitoneally.  These  animals  were  kept  under  observation  for 
several  months  and  showed  no  ill  effects  whatever  from  the  injec- 
tions of  filtrate. 

The  possibility  now  suggested  itself  that*  Bacillus  cholerse  sui» 
might  pass  through  the  walls  of  the  Pasteur-Chamberland  filter  in 
some  form  which  would  not  develop  in  vitro,  but  which  might  develop 
within  the  animal  body.  In  order  to  test  this  point  collodion  sacs 
were  prepared  according  to  the  methods  of  Grubbs  and  Francis," 
of  the  United  States  Public  Health  and  Marine-Hospital  Service, 
and  McCrae,''  of  the  Johns  Hopkins  Hospital.  Sacs  prepared 
according  to  each  of  these  methods  were  filled  with  the  filtrate  and 
inserted  into  the  abdominal  cavities  of  rabbits.  After  an  interval 
of  several  weeks  the  animals  were  killed  and  the  sacs  removed.  The 
filtrate  within  the  sacs  showed  no  apparent  clouding,  and  a  micro- 
scopic examination  of  the  contents  of  the  sacs  and  cultures  from 
the  same  failed  to  reveal  any  micro-organisms. 


EXPERIMENT  4. 


A  forty-eight-hour  bouillon  culture  (H  2199  S)  was  used  in  this 
experiment.  Filtration  was  made  through  a  Pasteur-Chamberland 
F  bougie.  In  this  experiment  and  in  those  which  follow  the  frac- 
tional method  of  filtration  was  used,  the  filtrates  being  collected  in 
separate  portions  in  small  sterile  Erlenmeyer  flasks,  which  were 
placed  in  the  incubator  as  soon  as  the  filtrations  were  completed. 

Table  showing  details  of  experiment  4. 


Portion 

Amount. 

Time  occupied 
in  passing  filter. 

Vacuimi. 

Result  of  incubation. 

I 

c.  c. 
75 
75 
75 
75 
35 
10 

5  minutes 

8  minutes 

10  minutes 

35  minutes  — 

3  hours 

Over  night 

Inches. 

21 
21 
21 
21 
21 
No  suction. 

Clouding  at  48  hours. 

II 

Do. 

Ill 

Clouding  at  7  days. 

IV 

Clouding  at  48  hours. 

v 

Remained  clear. 

VI 

Do. 

During  the  filtration  of  portions  I,  II,  and  III  the  bougie  was 
entirely  covered;  during  the  filtration  of  portions  IV,  V,  and  VI  it 
was  only  partly  covered.     Only  about  one-third   of  the  filter  was 

a  Grubbs,  S.  B.,  and  Francis,  Edward.  Laboratory  technique.  Collodion  sacs. 
U.  S.  Treasury  Department,  Public  Health  and  Marine-Hospital  Service,  Hygienic 
Laboratory,  Bulletin  7.     Washington,  1902. 

f>  McCrae,  John.  Notes  upon  the  agglutinations  obtained  by  intraperitoneal  inser- 
tion of  celloidin  capsules  containing  bacilli  and  upon  a  mode  of  preparing  such  cap- 
sules. Journal  "of  Experimental  Medicine,  vol.  5,  No.  C,  pp.  635-642.  New  York, 
Oct.  1,  1901. 


EXPERIMENTS   WITH   PASTEtTR-CHAMBERLAND   FILTERS.  17 

covered  with  culture  during  the  fihration  of  portion  V,  and  this, 
together  with  the  fact  that  the  pores  of  the  filter  liad  become  clogged, 
explains  the  length  of  time  required  for  this  portion  to  pass  the  filter. 

After  drawing  off  portion  V  the  suction  was  disconnected  and  the 
apparatus  left  in  place  overnight.  On  the  following  morning  it 
was  found  that  approximately  10  c.  c.  of  the  culture  had  passed  the 
filter,  and  this  portion  was  drawn  off  and  incubated  along  with  the 
other  fractions. 

Portions  I,  II,  and  IV  showed  clouding  at  forty-eight  hours. 
Microscopic  examinations  and  agar  plates  showed  in  each  case  pure 
cultures  of  a  micrococcus  corresponding  in  morphology  to  Staphy- 
lococcus 2>yogenes  albus,  and  probably  due  to  outside  contamination 
at  the  time  these  portions  were  drawn  off.  Guinea  pigs  weighing 
approximately  350  grams  each  were  injected  with  2  c.  c.  each  of 
these  portions  and  exhibited  no  ill  effects  from  the  injections. 

Portion  III  became  cloudy  after  one  week  in  the  incubator.  Micro- 
scopic exainination  and  agar  plates  revealed  a  pure  culture  of  a 
micrococcus  similar  to  that  found  in  portions  I,  II,  and  IV.  The 
injection  of  2  c.  c,  of  this  portion  into  a  guinea  pig  was  without  effect. 

Portion  V  was  kept  in  the  incubator  for  ten  days  and  remained 
perfectly  clear.  A  portion  of  this  filtrate  was  removed  at  the  end 
of  ten  days  by  means  of  a  sterile  pipette,  and  a  rabbit  weighing 
2,380  grams  was  given  a  subcutaneous  injection  of  10  c.  c.  without 
any  subsequent  ill  effects.  After  drawing  off  the  portion  for  the 
injecti(m  of  the  rabbit  the  remainder  of  the  fdtrate  was  inoculated 
with  culture  II  2199  S  in  order  to  see  whether  the  filtrate  would 
still  furnish  a  suitable  medium  for  the  growth  of  B.  cholerx  suis. 
The  inoculated  portion  was  replaced  in  the  incubator  and  showed 
well-marked  clouding  at  twenty-four  hours. 

Portion  VI  was  kept  in  the  incubator  for  ten  days  and  remained 
perfectly  clear.  It  was  then  inoculated  witli  culture  11  2199  S,  and 
showed  well-marked  clouding  at  twenty-four  hours,  thus  demon- 
strating that  B.  cholerx  suis  would  have  grown  in  this  filtrate  had 
it  passed  through  the  pores  of  the  filter. 

The  clouding  of  portions  I,  II,  III,  and  IV  in  this  experiment  was 
due  either  to  imperfect  sterilization  of  the  apparatus  or  to  outside 
contamination  at  the  time  these  portions  were  drawn  off.  That  the 
clouding  of  these  portions  was  not  due  to  the  passage  of  B.  cholerx 
suis  through  the  filter  is  shown  by  the  fact  that  guinea  pigs  inocu- 
lated with  these  portions  showed  no  ill  effects  from  the  inoculations. 

It  should  be  stateil  in  this  connection  that  the  experiments  described 
in  this  paper  were  conducted  in  a  large  general  bacteriological  work- 
room, where  the  conditions  were  more  or  less  favorable  for  the  outside 
contamination  of  the  iihrates,  owing  to  the  constant  passing  of  other 
workers  and  the  impossibility  of  avoiding  drafts  and  air  currents. 


18 


FILTRATION   EXPERIMENTS   WITH   B.   CHOLERiE   SUIS. 


EXPERIMENT  5. 

In  this  experiment  an  eighteen-hour  bouillon  culture  (II  2199  S) 
was  used.  The  reaction  of  the  bouillon  in  this  experiment  and 
those  which  follow  was  1  per  cent  acid.  Filtration  was  made  through 
a  Pasteur-Chaniberlaml  F  bougie.  The  fdtrate  was  collected  in 
separate  portions  in  small  sterile  Erlenmeyer  flasks,  which  were 
placed  in  the  incubator  as  soon  as  the  filtration  was  completed. 
The  bougie  was  kept  entirely  covered  with  culture  diu-ing  the  filtra/- 
tion.     The  amount  of  culture  fdtered  was  300  c.  c. 

Table  shoiving  details  of  experiment  5. 


Portion 

1  Time  oc- 

Amount. '  cupied  in 

j  filtration. 

Vaoinun. 

Kesult  of  incuba- 
tion. 

I 

II 

r.  r. 
75 
75 
75 
75 

Minutes. 
2 
2 
3 
4 

Inches. 

m 

20i 

Clouded. 
Remained  clear. 

UI 

Do. 

IV 

Do. 

Portion  I  showed  clouding  after  forty-eight  hours  in  the  incu- 
bator. Microscopic  examination  and  agar  plates  revealed  a  pure 
culture  of  a  large  spore-bearing  organism  resembling  Bacillus  sub- 
tilis.  A  guinea  pig  weighing  350  grams  was  injected  with  2  c.  c.  of 
this  portion  but  showed  no  ill  effects  from  the  injection.  The  organ- 
ism noted  in  this  flask  was  evidently  due  to  outside  contamination 
at  the  time  the  portion  was  drawn  off. 

Portions  II,  III,  and  IV  were  kept  in  the  incubator  for  two  weeks 
and  remained  perfectly  clear.  Two  portions  of  10  c.  c.  each  were 
then  removed  from  III  and  IV  by  means  of  sterile  pipettes  and 
injected  into  rabbits,  as  follows:  Rabbit  2224  (weight  2,620  grams) 
received  10  c.  c.  of  portion  III;  rabbit  2223  (weight  1,801  grams) 
received  10  c.  c.  of  portion  IV.  The  animals  were  kept  under  obser- 
vation for  several  months,  and  showed  no  ill  effects  whatever  from 
the  injections. 

After  withdrawing  portions  from  III  and  IV  for  the  injection  of 
rabbits,  the  remaining  portions  of  these  filtrates  were  inoculated 
with  B.  cholerse  suis  (culture  II  2199  S)  and  returned  to  the  incu- 
bator. After  twenty-four  hours  both  flasks  showed  well-marked 
clouding,  and  hanging-drop  preparations  and  agar  plates  showed 
pure  cultures  of  B.  cholerse  suis,  proving  that  the  absence  of  growth 
in  these  portions  after  filtration  was  not  due  to  exhaustion  of  the 
bouillon. 

EXPERIMENT  6. 

Two  fla.sks,  each  containing  400  c.  c.  of  beef  broth,  were  inoculated 
with  B.  cholerse  suis  (H  2199  S)  and  incubated  for  twenty-four  hours. 
The  contents  of  the  two  jQasks  were  then  poured  together  into  a 


EXPERIMENTS   WITH   PASTEUR-CHAMBERLAND    FILTERS. 


19 


large  balloon  flask  and  well  mixed.  The  object  of  this  was  to  give  a 
sufficient  quantity  of  culture  to  keep  the  bougie  covered  during  the 
entire  filtration.  Filtration  was  made  through  a  Pasteur-Chamber- 
land  F  bougie,  which  was  kept  entirely  covered  by  the  culture  through- 
out th'e  filtration.  The  filtrate  was  collected  in  separate  portions  of 
75  c.  c.  each  in  seven  flasks,  the  total  amount  of  culture  filtered 
being  525  c.  c. 

Table  shoming  details  of  experiment  6. 


Portion. 

Amount. 

Time  of 
filtration. 

Vacuum. 

Result  of  inculja- 
tion. 

I 

r.  r. 
75 
75 
75 
75 
75 
75 
75 

Minuteit. 
2 
3 

7 
9 
16 
19 
27 

Irtches. 

20 

20 

23-24 

23i-24 

24-25 

25 

25 

Clouded. 

II 

in 

Do. 

IV 

Do. 

V 

Do 

VI 

Do. 

vn 

Do. 

Portion  I  showed  sHght  clouding  at  forty-eight  hours,  and  a  micro- 
scopic examination  revealed  a  rather  long,  slender,  nonmotile  bacillus, 
apparently  in  pure  culture.  The  flask  was  returned  to  the  incubator 
and  allowed  to  remain  for  six  days,  at  the  end  of  which  time  it  showed 
well-marked,  uniform  clouding'.  Hanging-drop  preparations  and  agar 
plates  revealed  the  same  bacillus  that  had  been  noted  at  forty- 
eight  hours.  A  guinea  pig,  weighing  approximately  350  grams,  was 
injected  with  2  c.  c.  of  this  portion  after  incubation  for  six  days,  but 
showed  no  ill  effects  as  a  result  of  the  injection.  The  clouding  of  this 
portion  was  thus  proven  to  be  due  to  a  contaminating  organism  and 
not  to  B.  cholene  suis. 

Portions  II,  III,  IV,  V,  VI,  and  VII  were  incubated  for  twelve 
days,  and  remained  perfectly  clear.  Rabbits  were  injected  with 
portions  VI  and  VII  as  follows:  Ilabbit  2222  (weight  2,500  grams) 
received  10  c.  c.  of  portion  VI;  rabbit  2227  (weight  2,088  grams) 
received  10  c.  c.  of  portion  VII.  These  animals  were  kept  under 
observation  for  several  months,  and  remained  perfectly  well. 

After  removing  portions  for  the  injection  of  rabbits,  the  remaining 
portions  of  VI  and  VII  were  inoculated  with  B.  cholerse,  suis  (II  2199  S) 
and  replaced  in  the  incubator.  Both  flasks  showed  well-marked 
clouding  at  twenty-four  hours,  and  microscopic  preparations  and  agar 
plates  revealed  pure  cultures  of  B.  cholerse  suis.  This  was  done  in 
order  to  prove  that  the  bouillon  had  not  been  exhausted,  but  was 
still  a  suitable  medium  for  the  growth  of  B.  cholenx.  suis. 

KXPERIMENT  7. 

In  this  experiment  and  in  the  one  which  follows  Pasteur-Cham- 
berland  bougies  were  employed  which  had  already  been  u.sed  in  the 
preceding   experiments.     The   bougies   were   cleansed    according   to 


20 


FILTRATION   EXPERIMENTS   WITH   B.   CHOLERiE  SUIS. 


metliods  recommended  by  Lourens  before  being  used  the  second 
time.  Lourens,  it  should  be  stated,  did  not  use  new  filters  in  all  of 
his  experiments,  but  adopted  certain  metliods  for  cleansing  his  fiHere, 
and  the  two  experiments  which  follow  were  designed  with  the  view 
to  determining  whether  the  methods  which  he  adopted  for  cleansing 
his  filtere  could  have  had  any  effect  on  the  permeability  of  the  filters 
and  so  have  influenced  his  results. 

In  carrying  out  experiment  7  an  eighteen-hour  bouillon  culture 
of  B.  cholerse  suis  (H  2199  S)  was  used.  The  bougie  used  was  a  Pas- 
teur-Chamberland  F  which  had  been  used  once  before  in  experiment 
2.  Before  being  used  the  second  time  it  was  treated  by  the  method 
which  Lourens  first  used  for  cleansing  his  filters,  as  follows:  The  filter 
was  first  washed  with  cold  tap  water,  boiled  for  ten  minutes  in  1  per 
cent  hydrochloric  acid,  then  boiled  for  ten  minutes  in  a  3  per  cent 
sodium  carbonate  solution,  after  which  a  liter  or  more  of  cold  dis- 
tilled water  was  passed  through  the  filter  until  the  filtrate  showed 
only  a  faint  alkaline  reaction.  The  filter  was  sterilized  by  boiling 
for  one-half  hour  in  distilled  water.  The  filtrate  was  collected  in 
separate  portions.     The  amount  of  culture  filtered  was  750  c.  c. 

Table  showing  details  of  experiment  7. 


Portion. 

Amoimt. 

Time  of 
filtration. 

^'acuum. 

Result  of  incuba- 
tion. 

I 

C.C. 

75 

li  minutes. 

Inches. 
20} 

20  -18 
18-21 

21  -22} 

22} 
22}-21} 
21}-22 

22 
No  suction. 

II                                      

75      2    minutes. 
75      3    minutes. 

Do. 

Ill 

Do. 

IV 

75 
75 
75 
75 
75 
ISO 

6    minutes. 

9    minutes. 
12    minutes. 
16    minutes. 

IJ  hours — 
Overnight... 

Clouded. 

V               

Remained  clear. 

VI - 

Do. 

VII                       

Do. 

VIII 

Do. 

IX 

Do. 

During  the  filtration  of  the  first  seven  portions  the  bougie  was 
entirely  covered  with  culture;  during  the  filtration  of  portion  VIII 
the  bougie  was  partly  exposed.  After  portion  VIII  was  drawn  off 
the  vacuum  tubing  was  disconnected  from  the  side-arm  flask  and  the 
apparatus  left  in  place  overnight.  On  the  following  morning,  after 
standing  for  eighteen  hours,  150  c.  c.  of  filtrate  had  collected  in  the 
side-arm  flask.  This  portion,  which  passed  through  the  filter  during 
the  night,  was  then  drawn  off  and  placed  in  the  incubator,  together 
with  the  other  portions. 

Portions  I,  II,  III,  V,  VI,  VII,  VIII,  and  IX  remamed  perfectly 
clear  after  eleven  days  in  the  incubator.  Rabbits  were  then  injected 
subcutaneously  with  portions  VI  and  VII,  as  follows:  Rabbit  2225 
(weight  2,288  grams)  received  10  c.  c.  of  portion  VI;  rabbit  2226. 
(weight  2,185  grams)  received  10  c.  c.  of  portion  VII.  These  animals 
were  kept  under  observation  for  several  months,  and  showed  no  ill 
effects  from  the  inoculations. 


EXPERIMENTS   WITH    PASTEUR-CHAMBERLAND    FILTERS. 


21 


Careful  microscopic  examinations  were  made  of  several  of  these 
filtrates,  both  in  hanging-drop  and  in  smear  preparations  stained 
with  dilute  carbol-fuchsin,  and  small,  coccus-like  bodies  were  noted, 
but  cultures  from  the  same  portions  gave  negative  results. 

Portion  IV  showed  clouding  after  four  days,  and  agar  plates  revealed 
a  pure  culture  of  a  medium-sized  micrococcus  corresponding  in  mor- 
phology with  S.  pyogenes  alhus.  A  guinea  pig  was  inoculated  with 
2  c.  c.  of  this  portion  after  clouding  had  developed,  and  showed  no 
ill  effects  from  the  inoculation.  The  growth  in  this  flask  was  evi- 
dently due  to  outside  contamination  when  the  portion  was  drawn  oil. 
This  is  shown  by  the  fact  that  -all  of  the  portions  drawn  off  before 
this  one,  as  well  as  all  of  those  drawn  off  after  it,  were  sterile. 

Several  of  the  filtrates  after  being  incubated  for  eleven  days  with 
negative  results  were  inoculated  with  B.  cholerx  suis  and  gave  well- 
marked  clouding  at  twenty-fouT  hours,  showing  that  the  culture 
medium  was  still  suitable  for  the  growth  of  B.  cholerse  suis. 

EXPERIMENT  8. 

The  culture  used  in  this  experiment  was  a  twenty-four-hour  bouillon 
of  B.  cholerse  suis  (H  2 1 99  S) ,  Filtration  was  made  through  a  Pasteur- 
Chamberland  F  bougie  which  had  been  used  once  in  ex])eriment  6. 
The  filter  was  cleansed  according  to  the  method  finally  adopted  by 
Lourens  as  yielding  the  most  satisfactory  results.  Following  this 
method,  the  outside  of  the  filter  was  first  washed  with  cold  tap  water; 
a  liter  of  cold  distilled  water  was  then  passed  through  the  filter;  next 
a  liter  of  ])otassium  permanganate  solution  (1  gm.  KMnO^,  6.5  gins. 
HCl,  1,000  c.  c.  water)  was  drawn  through  the  filter;  following  the 
permanganate,  a  like  amount  of  oxalic-acid  solution  (10  gms.  oxalic 
acid  to  1,000  c.  c.  water)  was  passed  through  the  filter;  hot  water 
was  next  drawn  through  the  filter  until  the  filtrate  was  acid-free,  and 
finally  a  liter  of  cold  distilled  water  was  passed  through.  Before 
being  used  the  filter  was  sterilized  in  the  usual  manner  by  boiling  for 
one-half  hour  in  distilled  water.  The  filtrate  was  collected  in  separate 
portions,  the  total  amount  of  culture  filtered  being  525  c.  c.  During 
the  filtration  the  entire  filter  was  covered  with  tlu^  culture. 

Table  shounng  details  of  experiment  S. 


Portion. 

Am 

junt. 
c. 

Tlmo  of 
filtration. 

MintUen. 

Vaciiiiiii. 
Inches. 

Re.siilt  of  incuba- 
tion. 

c. 

I 

7.5 

24 

23* 

Remained  clear. 

H 

75 

« 

•m 

Do. 

HI 

75 

9 

23J 

Do. 

IV 

75 

1.3 

23i 

Do. 

V 

1 

75 

22 

23J-17 

Do. 

VI 

1 

75 

30 

8 

Do. 

VII 

••-. 1 

75 

40 

»-10 

Do. 

22 


FILTRATION   EXPERIMENTS   WITH   B.    CHOLERA  SUIS. 


Portions  I  to  VII,  inclusive,  remained  perfectly  clear  after  ten  days 
in  the  incubator,  at  the  end  of  which  time  two  rabbits  were  injected 
subcutaneously  with  portions  VI  and  VII,  as  follows:  Rabbit  2118 
(weight  1,875  grams)  received  10  c.  c.  of  portion  VI;  rabbit  2119 
(weight  1,892  grams)  received  10  c.  c.  of  portion  VII.  The  animals 
were  kept  under  observation  for  several  months,  but  showed  no  ill 
effects  from  the  injections.  After  withdrawing  sufficient  quantities 
of  portions  VI  and  VII  for  the  injection  of  the  rabbits,  the  remainders 
of  these  portions  were  inoculated  with  B.  cholerae.  suis  and  showed 
well-marked  clouding  after  twenty-four  hours. 

Portions  I,  II,  III,  IV,  and  V  were  left  in  the  incubator  for  six 
weeks  and  remained  perfectly  clear.  A  microscopic  examination  of 
several  of  these  portions  revealed  small  bodies  like  those  noted  in 
experiment  7,  but  cultures  from  these  portions  yielded  negative  results, 

EXPERIMENT  9. 

A  twenty-six  hour  bouillon  culture  of  B.  cholerse  suis  (H  2450  S) 
was  used  in  this  experiment.  The  filter  used  was  a  new  Pasteur- 
Chamberland  F  bougie.  The  filtrate  was  collected  in  seven  portions 
of  50  c.  c.  each,  the  total  amount  of  culture  filtered  being  350  c.  c. 
The  bougie  was  kept  covered  with  culture  during  the  entire  filtration. 


Table  showing  details  of  experiment . 

9. 

Portion. 

Amount. 

Time  of 
filtration. 

Vacuum. 

Result  of  incuba- 
tion. 

I      

c.  c. 
50 
50 
50 
50 
50 
50 
50 

40  seconds 

1^  minutes 

2  minutes 

4J  minutes 

0  minutes 

7i  minutes 

17  minutes 

Inches. 
23 
23 
23 
23 
20 
20 
20 

Remained  clear. 

II -. 

Do. 

in 

Do. 

IV 

Do. 

v 

Do. 

VI 

Do. 

vn 

Do. 

The  filtrates  were  placed  in  the  incubator  immediately  after  filtra- 
tion and  showed  no  perceptible  clouding  after  prolonged  incubation. 

Portions  I,  II,  VI,  and  VII  were  left  in  the  incubator  for  six  weeks 
and  remained  perfectly  clear.  Cultures  were  made  from  these  por- 
tions on  various  laboratory  media,  including  Martin's  serum-broth, 
with  negative  results. 

Portion  III  was  removed  from  the  incubator  at  the  end  of  two 
weeks  and  was  found  to  be  perfectly  clear.  This  portion  was  sub- 
jected to  a  careful  miscroscopic  examination.  In  hanging-drop 
preparations  small  bodies  having  somewhat  the  appearance  of 
micrococci  were  noticed.  In  preparations  stained  with  dilute  carbol- 
fuchsin  which  had  been  carefully  filtered  through  double  filters  to 
remove  all  dirt  and  granules,  small  bodies  were  also  noted;  these 
bodies  varied  in  size  from  minute  points  to  bodies  0.6  pi  in  diameter. 
No  bacilli  were  noted  in  any  of  the  preparations. 


EXPERIMENTS   WITH    PASTEUR-CHAMBERLAND    FILTERS.  23 

Cultures  were  made  from  this  portion  as  follows:  Two  tubes  of 
neutral  agar,  two  tubes  of  neutral  beef  broth,  and  two  tubes  of 
Martin's  bouillon  with  normal  hog  serum  added  were  inoculated 
with  1  c.  c.  each,  but  none  of  these  cultures  showed  any  growth 
after  two  weeks'  incubation.  Martin's  bouillon,  with  the  addition  of 
serum,  was  the  medium  upon  which  Nocard  and  Roux"  in  1898  first 
succeeded  in  cultivating  outside  of  the  animal  body  the  organism  of 
bovine  pleuro-pneumonia,  an  organism  which  up  to  that  time  had 
resisted  all  attempts  at  cultivation  and  had  been  classed  among  the 
ultravisible  viruses.  Nocard  and  Roux  employed  ^lartin's  bouillon 
with  the  addition  of  beef  or  rabbit  serum,  whereas  in  the  present  ex- 
periments hog  serum  was  used  as  affording  a  more  suitable  medium 
for  the  possible  growth  of  any  living  particles  or  bodies  which  might 
have  passed  through  the  pores  of  the  filters. 

Two  hogs  weighing  from  30  to  40  pounds  each  were  injected  with 
portion  III  as  follows:  Hog  2351  received  an  intravenous  injection 
in  ear  vein  of  10  c.  c.  of  portion  III;  hog  2352  received  a  subcutaneous 
injection  in  groin  of  20  c.  c.  of  portion  III.  These  animals  were  kept 
under  observation  for  several  months  and  remained  perfectly  well. 

In  explaining  his  failure  to  produce  hog  cholera  by  the  subcutaneous 
injection  of  cultures  of  B.  cholerse  suis  and  the  high  degree  of  virulence 
which  the  blood  of  animals  sick  of  hog  cholera  exhibits  when  injected 
subcutaneously,  Theobald  Smith''  advanced  the  theory  that  the 
blood  coagulates  in  the  connective  tissues  and  serves  as  a  food  for  the 
bacilli  which  it  incloses  and  at  the  same  time  protects  them  against 
the  action  of  the  leucocytes.  With  this  theory  in  mind,  and  in  order 
to  determine  whether  the  coccus-like  bodies  noted  in  this  filtrate  pos- 
sessed any  significance — that  is,  whether  they  were  capable  of  further 
development  in  the  animal  body — the  following  experiment  was  car- 
ried out:  Ten  cubic  centimeters  of  blood  was  drawn  from  the  tail  of 
a  healthy  hog  under  aseptic  conditions  and  collected  in  a  large  sterile 
test  tube,  the  interior  of  which  had  received  a  coating  of  sterile  olive 
oil  to  prevent  coagulation.  To  this  blood  was  immediately  added  10 
c.  c.  of  portion  III.  The  mixture  of  blood  and  filtrate  was  then 
drawn  up  into  a  sterile  syringe  and  at  once  injected  into  the  groin  of  a 
healthy  hog  (No.  2354)  before  coagulation  had  time  to  take  place. 
Now,  if  the  coccus-like  bodies  noted  in  this  filtrate  had  been  caj)able 
of  developing  into  bacillary  forms,  as  Lourons  claimed  for  tiiosc  which 
he  observed,  they  were  certainly  afforded  in  this  exj)eriment,  accord- 
ing to  Smith's  theory,  a  good  opportunity  to  develop  within  the 
animal  body.  This  hog,  however,  was  kept  under  observation  for 
several  months  and  remained  perfectly  well. 

a  Nocard,  E.  I.  E.,  and  Roux.  Le  microbe  de  la  p6ripneumonie.  Anuales  de 
rin.stitut  Pa-^tcur,  tome  12,  No.  4,  pp.  240-202,  Pari.s,  Apr.  25,  1908. 

6  Hog  Cholera:  Its  History,  Nature,  and  Treatment.  United  States  Department  of 
Agriculture,  1889. 


24 


FILTRATION   EXPERIMENTS  WITH  B.   CHOLERiE   SUIS. 


Portion  IV  was  removed  from  the  incubator  at  the  end  of  three 
weeks.  Hanging-drop  and  smear  preparations  stained  with  dilute 
carbol-fuchsin  showed  bodies  similar  to  those  noted  in  portion  III,  but 
no  bacilli.  Two  rabbits,  weighing  approximately  2,000  grams  each, 
were  injected  as  follows:  Rabbit  2306  received  an  intravenous  in- 
jection of  3  c.  c.  of  portion  IV;  rabbit  2307  received  a  subcutaneous 
injection  of  10  c.  c.  of  portion  IV.  Both  animals  remained  well  and 
showed  no  ill  effects  as  a  result  of  the  injections. 

Portion  V  was  removed  from  the  incubator  at  the  end  of  four  weeks. 
Microscopic  examination  revealed  the  same  bodies  noted  in  portions  III 
and  IV,  but  there  had  been  no  apparent  multiplication  of  these  bodies 
and  no  bacilli  were  observed.  Two  rabbits,  weighing  approximately 
2,300  grams  each,  were  injected  as  follows:  Rabbit  2301  received 
an  intravenous  injection  of  3  c.  c.  of  portion  V;  rabbit  2308  received 
a  subcutaneous  injection  of  10  c.  c.  of  portion  V.  Both  animals  re- 
mained well,  showing  no  ill  effects  whatever  from  the  injections. 

EXPERIMENT  10. 

In  connection  with  his  culture  experiments  Lourens  states  that  the 
addition  of  an  equal  quantity  of  serum  to  bouillon  furnishes  a  fluid 
which  considerably  increases  the  permeability  of  filters  of  the  Berke- 
feld  and  Pasteur-Chamberland  types.  In  order  to  test  the  correct- 
ness of  this  statement  with  regard  to  filters  of  the  Pasteur-Cl^amber- 
land  type  the  following  experiment  was  carried  out : 

A  flask  containing  250  c.  c.  of  bouillon  was  inoculated  with  B.  chol- 
erse  suis  (H  2228  S)  and  incubated  for  forty-four  hours.  An  equal 
amount  (250  c.  c.)  of  horse  serum  was  then  added  to  the  bouillon  cul- 
ture and  the  mixture  thoroughly  shaken.  Filtration  was  made 
through  a  new  Pasteur-Chamberland  F  bougie.  The  filtrate  was  col- 
lected in  seven  portions  of  70  c.  c.  each,  the  total  amount  of  culture 
filtered  being  490  c.  c. 

Table  showing  details  of  experiment  10. 


Portion. 

Amount. 

Time  of 
filtration. 

Vacuum. 

Result  of  incubation. 

I 

c.  c. 
70 
70 
70 
70 
70 
70 
70 

Minutes. 
1 

1 

i' 

7 
27 

Inches. 
28 
28 
28 
28 
28 
28 
28 

Filtrate  free  from  B.  cholerx  suis. ' 

U 

Do. 

ni 

Do. 

IV 

Do. 

V 

Do. 

VI 

Do. 

vu 

Do. 

After  twenty-four  hours  in  the  incubator  the  filtrates  all  showed  a 
slight  opalescence,  but  there  was  no  apparent  clouding  of  the  medium. 
After  one  week  in  the  incubator  the  opalescence  noted  at  the  end  of 
twenty-four  hours  had  not  increased  and  there  was  still  no  clouding 
of  the  filtrates. 


SUMMARY  OF  PASTEUR-CHAMBERLAND  FILTRATIONS. 


25 


Cultures  were  made  from  portions  I,  II,  V,  VI,  and  VII  on  various 
laboratory  media,  including  Martin's  serum-broth,  with  negative  re- 
sults. 

Portions  III  and  IV  were  removed  from  the  incubator  at  the  end  of 
one  week  and  subjected  to  a  careful  microscopic  examination.  Smear 
preparations  stained  with  dilute  carbol-fuchsin  showed  a  finely  granu- 
lar material  and  occasional  smjall  coccus-like  bodies  like  those 
noted  in  experiment  9.  Cultures  were  made  from  each  of  these  por- 
tions by  adding  10  c.  c.  to  flasks  containing  400  c.  c.  each  of  beef  broth. 
Rabbits  were  then  given  subcutaneous  injections  as  follows:  Rabbit 
2428  (weight  2,160  grams)  received  10  c.  c.  of  portion  III;  rabbit  2429 
(weight  2,935  grams)  received  10  c.  c.  of  portion  IV. 

After  the  removal  by  means  of  sterile  pipettes  of  the  portions  used 
for  the  inoculation  of  cultures  and  rabbits,  portions  III  and  IV  were 
then  inoculated  with  B.  choleras  suis  (H  2228  S)  and  replaced  in  the 
incubator.  Both  portions  showed  well-marked  clouding  at  the  end 
of  twenty-four  hours,  and  microscopic  examination  revealed  actively 
motile  forms  of  B.  cholerse.  suis,  thus  proving  that  the  medium  was  not 
unfavorable  to  the  growth  of  the  bacillus. 

The  two  flasks  of  bouillon  which  were  inoculated  with  10  c.  c.  each 
of  portions  III  and  IV  failed  to  show  any  growth  upon  prolonged  in- 
cubation, and  the  two  rabbits  inoculated  with  like  amounts  of  the 
same  portions  showed  no  ill  effects  as  a  result  of  the  inoculations. 

The  opalescence  noted  in  the  filtrates  was  evidently  due  to  a  slight 
precipitation  of  albuminous  material  from  the  blood  serum. 

This  experiment  would  show  that  in  the  case  of  the  Pasteur-Cham- 
berland  filter  the  addition  of  serum  to  the  material  filtered  does  not 
affect  the  permeability  of  the  filter. 

SUMMARY  OF  EXPERIMENTS  WITH  PASTEUR-CHAMBERLAND  FILTERS, 

The  results  of  the  experiments  with  Pasteur-Chamberland  filters 
are  summarized  in  the  following  table : 

Summary  of  Pasteur-  Chamberland  fUtrations. 


No.  of 
experiment. 

Date  l)egun. 

(^ture. 

Grade  of 
filter. 

Amount 
of  culture 
filtered. 

Time 
occu- 
pied 
In  fil- 
tration. 

Vacu- 
um. 

Result  of 
Intubation. 

Number. 

Age. 

1 

Feb.  24,1904 

Mar.     2, 1904 
June     6, 1905 
Nov.  29, 1907 
Nov.  30, 1907 
Dec.     3, 1907 

Gp  3998  8. 
...do.... 

24  hours  . . 
do 

F 

F  and  B 
B 
F 
F 

F 
F 
F 
K 
F 

c.  c. 
200 

200 
100 

0  335 
300 
525 

a(XX) 

h.    VI. 

35 

45 

37 

3    58 

11 

1  23 

2  4A 

Inches. 
23 

21-22 
20 
21 
20J 

20-25 

18-22J 
8-23J 

20-2;{ 
28 

B.   cholerx 

2 

«««■«  absent. 
Do. 

3 

(ip  4092  S. 
U2199S.. 
do 

fin 

7  days  

2  days  

18hours... 
24hours... 

Do. 

4 

Do. 

5 

Do. 

6 

Do. 

7 

Dec.     5,1907       ...do..   . 

Do. 

8 

Dec.     7,1907    do  ... .,  24hours. . . 

June  12,1908     11  24.'»  S  ..    20hours. .. 

Aug.    24,1908      112-i^.S         44  hours 

525  I    2      2i 
350            39 

490  1    I    '^n 

Do. 

9 

Do. 

10 

Do. 

oThe  portions  which  filtered  through  overnight  under  atmospheric  pressure  are  not  Included  in  this 
table. 


26  FILTRATION   EXPERIMENTS   WITH  B.   CHOLERA  SUIS. 

DISCUSSION    OF    PASTEUR-CHAMBERLAND   FILTRATIONS. 

In  the  experiments  just  described,  nine  Pasteiu'-Chamberland  filters 
were  tested  with  bouillon  cultures  of  B.  cholerse.  suis  under  an  average 
vacuum  of  20  to  25  inches.  Five  different  strains  of  B.  cholerse  suis 
were  used,  and  the  cultures  filtered  varied  in  age  from  eighteen  hours 
to  seven  days.  The  amount  of  culture  filtered  varied  from  100  to 
750  c.  c,  and  in  not  a. single  instance  coidd  B.  cliolerx  suis  be  detected 
in  the  filtrates. 

In  testing  the  filtrates  for  B.  cholersB  suis  one  or  more,  sometimes 
all,  of  the  following  methods  were  used:  (1)  Incubation  of  the  fil- 
trate, either  entire  or  in  fractions,  for  a  considerable  period  of  time; 
(2)  the  injection  of  large  amounts  of  the  filtrate  into  susceptible 
animals;  (3)  the  introduction  of  collodion  sacs  containing  filtrate  into 
the  abdominal  cavities  of  susceptible  animals;  (4)  microscopic  ex- 
amination, in  both  hanging-drop  and  stained  preparations;  (5)  sub- 
cultures on  various  media  favorable  to  the  growth  of  B.  cholerse  suis. 
By  none  of  these  methods,  however,  could  B.  cholerse  suis  be  detected 
in  any  of  the  filtrates,  and  the  conclusion  therefore  seems  justified 
that  the  organisms  were  completely  restrained  by  the  filters. 

In  view  of  the  fact  that  all  of  the  nine  filters  tested  proved  equally 
efficient  in  preventing  the  passage  of  B.  cholerse  suis,  we  ma}^  further 
conclude  that  the  Pasteur-Chamberland  filter  may  be  depended  on 
with  safety  to  furnish  as  much  as  500  to  600  c.  c.  of  bacteria-free 
filtrates  from  bouillon  cultures  or  correspondingly  heavy  suspensions 
of  organisms  approximating  in  size  to  B.  cholerse  suis.  As  it  has  been 
shown  that  bacteria  are  capable  of  growing  through  the  walls  of 
bacterial  filters,  it  is  best  to  limit  the  duration  of  the  filtration  to 
two  or  three  hours.  In  two  of  the  experiments  recorded  in  tliis 
paper  (Chamberland  experiments  4  and  7)  the  apparatus  was  left 
in  place  and  the  portions  which  passed  through  the  filters  overnight 
were  collected  and  incubated.  These  portions  remained  sterile, 
showing  that  in  two  instances  at  least  B.  cholerse  suis  did  not  grow 
through  the  walls  of  the  Chamberland  F  filter  in  sixteen  hours.  Just 
how  long  it  will  require  for  different  bacteria  to  grow  through  the 
Chamberland  filter  is  a  point  which  has  not  been  fully  investigated. 

Although  it  does  not  seem  probable  in  view  of  the  perfect  efhciency 
exhibited  by  the  Pasteur-Chamberland  filters  in  the  experiments 
just  described,  it  is  nevertheless  possible  that  an  imperfect  bougie 
may  occasionally  go  out  on  the  market,  and  for  this  reason  it  is  im- 
portant in  all  filtration  experiments  where  bacteria-free  filtrates  are 
desired,  even  where  the  Pasteur-Chamberland  filter  is  used,  that  all 
filtrates  as  well  as  filters  be  carefully  tested. 

Even  if  the  experiments  described  in  BuUetin  72  of  this  Bureau, 
previously  mentioned,  to  which  Lourens  takes  exception,  had  not 


DISCUSSION   OF   PASTEUR-CHAMBERLAND   FILTRATIONS.  27 

been  sufficiently  and  carefully  checked  at  the  time  they  were  made, 
the  experiments  with  Pasteur-Chamberland  filters  described  in  the 
present  paper  would  go  far  to  prove  that  the  filtrates  employed  in 
Bulletin  72  were  absolutely  free  from  B.  cholerse  suis,  for  in  nearly 
all  of  the  filtration  experiments  described  in  that  bulletin  the  Pasteur- 
Chamberland  type  of  filter  was  used  almost  exclusively,  a  new  filter 
being  used  for  each  experiment,  and  the  amount  of  fdtrate  collected 
never  exceeded  500  cubic  centimeters. 

Lourens,  however,  criticises  the  filtration  experiments  described  in 
Bulletin  72  on  the  ground  that  in  testing  the  sterility  of  the  filtrates 
cultures  were  made  from  the  filtrates  immediately  after  filtration. 
He  claims  that  his  experiments  show  that  cultures  made  from  filtrates 
immediatel}"  after  filtration  will  almost  always  remain  sterile,  even 
though  they  be  kept  at  incubator  temperature  for  several  weeks. 
This  he  explains  on  the  ground  that  there  may  be  so  few  organisms 
in  the  filtrate  that  they  are  missed  in  taking  out  portions  for  cultures. 
In  making  this  criticism  he  apparently  leaves  out  of  consideration  a 
point  upon  which  he  lays  great  emphasis  in  his  conclusions,  namely, 
the  formation  of  granules  sufficiently  small  to  pass  through  the  walls 
of  a  porcelain  filter  and  capable  of  developing  in  the  filtrates  into  B. 
cholerae,  suis.  Now,  if  these  granules  be  sufficiently  small  to  pass 
through  the  walls  of  a  porcelain  filter,  they  should  be  present  in  the 
filtrates  in  considerable  numbers  and  could  hardly  be  missed  in  taking 
out  portions  for  cultures.  But  aside  from  this  apparent  contradic- 
tion, we  do  not  consider  Lourens's  criticism  well  founded,  for  as 
previously  stated,  repeated  tests  of  the  filtrates  described  in  Bulletin 
72  showed  that  cultures  made  with  several  times  the  amount  of  fil- 
trate used  for  the  inoculation  of  hogs  did  not  show  B.  choler-x  suis  in 
a  single  instance,  and  guinea  pigs  and  rabbits  were  unaffected  by 
amounts  sufficient  to  cause  the  death  of  hogs. 

In  the  experiments  described  in  the  present  paper  the  filtrates  were 
held  at  incubator  temperature  for  from  one  to  two  weeks  before  they 
were  tested  on  animals,  and  supposing  that  only  a  very  few  organisms 
had  passed  through  the  filters  into  the  filtrates  these  would  certainly 
have  had  ample  time  to  develop  before  the  filtrates  were  tested. 
Where  the  entire  filtrate  was  held  at  incubator  temperature  for  a 
considerable  time,  as  just  explained,  it  did  not  seem  necessary  to 
make  cultures  from  the  filtrates  in  every  instance,  as  it  was  repeat- 
edly proven  by  subsequent  inoculation  that  tlie  filtrates  themselves 
afforded  a  suitable  medium  for  the  growth  of  B.  cholersR  suis  had  this 
organism  succeeded  in  passing  through  the  wails  of  tiie  filter. 

Pasteur-Chamberland  filters,  which  were  subjected  to  a  single 
cleansing  according  to  the  methods  recommended  by  Lourens,  were 
apparently  unimpaired  in  efficiency,  Init  it  does  not  seem  unlikely 
that  repeated  cleausiugs  by  these  methods  might  in  time  affec 


i  methods  might  in  time  affect  their-jDAU 

oADiOW  MEDICAL  DBRAh 

,  OQ  ANGELES.  CAL 


28  FILTRATION   EXPERIMENTS   WITH   B.   CHOLERA  SUIS. 

pores  of  the  filter  and  render  it  more  permeable.  Indeed,  it  is  hard 
to  explain  the  results  of  I^ourens's  Pasteur-Chamberland  filtrations 
on  any  other  ground  unless  it  be  that  he  used  excessively  high  pres- 
sures in  carrying  out  his  filtrations.  Lourens  describes  six  experi- 
ments with  bouillon  cultures  of  B.  cholerse  stiis  in  which  Pasteur- 
Chamberland  filters  were  used,  and  in  four  out  of  the  six  experiments 
B.  cJiolersR  suis  appeared  in  the  filtrates.  In  none  of  these  six  experi- 
ments does  he  appear  to  have  used  new  filters,  nor  does  he  state  how 
often  his  filtere  had  been  previously  used  and  cleansed,  but  ])resum- 
ably  they  had  been  used  a  number  of  times.  Lourens  also  fails  to 
describe  the  manner  in  which  he  arranged  his  filters,  whether  for  air 
pressure  or  vacuum,  and  nowhere  in  the  course  of  his  article  does  he 
record  the  amount  of  pressure  or  vacuum  employed.  It  would  appear, 
however,  from  the  brief  paragraph  which  he  devotes  to  the  technique 
of  his  filtration  experiments  that  he  made  use  of  pressure  in  carrying 
out  his  filtrations,  his  filters  being  so  arranged  that  air  pressure  could 
be  applied  to  the  fluid  surrounding  the  filter  and  the  fluid  thus  forced 
through  the  walls  of  the  filter  instead  of  being  drawn  through  by 
means  of  suction.  That  he  employed  considerable  pressure  in  carry- 
ing out  his  experiments  is  indicated  from  his  statement  that  he  expe- 
rienced no  difficulty  in  passing  500  c.  c.  of  undiluted  blood  serum 
through  a  Berkefeld  filter  (size  of  filter  not  stated)  in  fifteen  minutes 
or  less,  and  that  it  required  but  little  longer  to  pass  the  same  amount 
of  serum  through  a  Chamberland  F  filter. 

As  this  last  statement  in  regard  to  the  filtration  of  blood  serum  is 
so  at  variance  with  our  own  experience  and  the  experience  of  other 
investigators  who  have  found  that  blood  serum  filters  with  consider- 
able difficulty,  we  have  tested  a  number  of  Berkefeld  and  Chamberland 
filters  with  distilled  water  and  blood  serum,  in  order  to  determine 
their  approximate  rates  of  filtration. 

RATE    OF  FILTRATION   OF  BERKEFELD   AND   PASTEUR- 
CHAMBERLAND    FILTERS. 

Employing  a  vacuum  of  29  inches,  it  required  about  fifteen  minutes 
to  pass  500  c.  c.  of  distilled  water  through  a  No.  7  Berkefeld  labora- 
tory cylinder,  about  ten  minutes  to  pass  the  same  amount  through 
a  Chamberland  F  filter,  and  from  fifteen  to  twenty  minutes  to  pass 
a  like  amount  through  a  Chamberland  B  filter.  With  clear,  limpid 
hog  serum,  which  was  first  passed  through  a  large  No.  2  Berkefeld 
cylinder,  in  order  to  free  it  from  all  red  blood  cells,  it  required  over 
three  hours  to  pass  500  c.  c.  through  a  Berkefeld  laboratory  cylinder 
No.  7  under  a  vacuum  of  29  inches  and  over  four  hours  to  pass  300  c.  c. 
through  a  Chamberland  F  filter  under  the  same  vacuum.  In  the  case 
of  both  filters  the  filtration  of  the  blood  serum  proceeded  with  pro- 
gressive slowness,  as  was  shown  by  collecting  the  filtrates  in  sepa- 
rate portions  of  100  c.  c.  each.     In  the  case  of  the  Berkefeld  filter 


RATE   OF   FILTRATION   WITH  DIFFERENT   FILTERS.  29 

the  first  100  c.  c.  of  serum  passed  the  filter  in  six  minutes,  the  sec- 
ond in  nine  minutes,  the  third  in  twelve,  and  the  fourth  in  tliirty 
minutes,  whereas  the  fifth  portion  of  100  c.  c.  required  two  and  one- 
half  hours  to  pass  the  filter,  at  the  end  of  which  time  the  filter  had 
virtually  ceased  to  act.  In  the  case  of  the  Chamberland  filter  the 
first  100  c.  c.  of  serum  passed  the  filter  in  fifteen  minutes,  the  second 
in  one  hour,  while  the  third  portion  of  100  c.  c.  required  three  hours 
to  pass  the  filter,  at  the  end  of  which  time  the  filter  had  become 
completely  clogged  and  filtration  had  practically  ceased. 

In  view  of  these  results,  it  is  difficult  to  see  how  Lourens  succeeded 
in  passing  500  c.  c.  of  blood  serum  through  a  Chamberland  F  filter 
in  fifteen  minutes,  unless  he  used  very  high  pressure,  and  if  he  used 
such  pressure  it  is  conceivable  that  in  his  filtration  experiments  the 
clouding  of  his  filtrates  from  B.  cholerse,  suis  was  due  to  the  fact  that 
he  actually  forced  the  organisms  themselves  through  the  walls  of  his 
filters. 

GRANULE  FORMATION  IN  CULTURES  OF  B.  CHOLERA  SUIS. 

With  regard  to  the  presence  of  granules  in  cultures  of  B.  cJiolerse 
suis,  Lourens  states  that  only  certain  strains  possess  this  property  of 
breaking  up  into  granules,  but  in  those  strains  which  are  character- 
ized by  granule  formation  the  peculiarity  is  a  fixed  one  and  persists 
after  the  organism  has  been  cultivated  on  the  different  culture  media 
and  also  after  it  has  been  passed  through  the  animal  body.  He  states 
that  in  his  experiments  only  those  cultures  which  were  characterized 
by  granule  formation  and  polar  staining  passed  through  the  filters, 
his  theory  being  that  the  granules  are  sufficiently  small  to  pass 
through  the  pores  of  the  filters,  and  after  passing  through  the  filters 
these  granules  are  capable  of  developing  in  the  filtrates  into  the 
characteristic  bacillary  forms.  He  describes  the  granule  formation 
as  giving  rise  to  coccus-like  forms  which  may  have  the  appearance 
of  constricted  bacilli  or  cocci  lying  in  close  juxtaposition,  and  now 
and  then  as  three  coccus-like  bodies  lying  free  or  surrounded  by  a 
zone  resembling  a  capsule. 

Three  of  the  five  cultures  used  in  the  experiments  described  in  the 
present  paper  were  carefully  examined  for  granules  like  those  de- 
scribed by  Lourens.  The  stock  cultures  of  two  of  the  strains  experi- 
mented with  were  unfortunately  lost  in  moving  the  laboratory  and 
could  not  be  examined  for  granules.  In  examining  the  cultures  for 
granules,  smear  preparations  were  made  from  bouillon  cultures  and 
from  the  water  of  condensation  of  agar  cultures.  The  films  were 
carefully  fixed  by  means  of  formalin  and  methyl  alcohol  without  the 
aid  of  heat,  so  as  to  avoid  any  possible  distortion  from  overheating, 
and  were  stained  with  dilute  carbol-fiichsin,  as  reconnnended  by  Lou- 
rens.    In  the  three  cultures  examined  polar  staining  was  obsei-vecl 


30  FILTRATION   EXPERIMENTS   WITH  B.   CHOLER-E  SUIS. 

in  all  and  granular  forms  were  noted  which  corresponded  in  every 
particular  with  those  described  and  pictured  by  Lourens.  We  must 
admit,  therefore,  the  correctness  of  Lourens's  observation  as  to  the 
formation  of  granules  in  cultures  of  B.  cholerx  suis,  and  it  is  also 
possible  that  these  granules  may  pass  through  the  pores  of  an  unglazed 
porcelain  filter;  but  in  view  of  the  results  of  our  Pasteur-Chamberland 
filtrations,  where  the  filtrates  were  proven  in  every  instance  to  be  free 
from  B.  cholerse  suis,  although  at  least  three  of  the  cultures  experi- 
mented with  showed  granule  formation,  we  must  conclude  that  the 
granules  upon  which  Lourens  lays  so  much  stress  are  incapable  of 
developing  into  the  characteristic  bacillary  forms  of  B.  cholerse  suis 
and  that  they  possess  no  significance  in  filtration  experiments  with 
this  organism. 

In  our  experiments  with  the  Berkefeld  filter,  where  B.  choleras,  suis 
developed  in  the  filtrates,  we  believe  that  the  organisms  themselves 
passed  through  the  walls  of  the  filter. 

STJMMABY  OF  RESULTS. 

When  bouillon  cultures  of  B.  cholerse  suis  were  filtered  through  the 
smaller  Berkefeld  laboratory  filters  it  was  found  that  after  a  time — 
that  is,  after  a  certain  amount  of  culture  had  been  filtered — the  organ- 
isms appeared  in  the  filtrates.  With  a  vacuum  of  20  to  25  inches  of 
mercury  not  more  than  100  c.  c.  of  bacteria-free  filtrate  could  be 
obtained  with  these  filters.  When  bouillon  cultures  of  B.  cholerse  suis 
were  filtered  through  Pasteur-Chamberland  filters  (F  and  B),  the 
organisms  did  not  appear  in  the  filtrates  in  a  single  instance,  although 
as  much  as  600  c.  c.  of  culture  was  filtered  in  one  instance.  With  a 
vacuum  of  20  to  25  inches  of  mercury,  the  Pasteur-Chamberland  filters 
(F  and  B)  can  be  depended  on  to  furnish  from  500  to  600  c.  c.  of 
bacteria-free  filtrate  from  bouillon  cultures  of  B.  cholerse  suis  when  the 
time  consumed  in  filtration  does  not  occupy  more  than  two  hours. 

Beef  broth  or  bouillon  is  apparently  unaltered  by  passage  through 
a  Berkefeld  or  Pasteur-Chamberland  filter,  and  the  absence  of  growth 
in  filtrates  from  bouillon  cultures  of  B.  cholerse  suis  can  not  be  explained 
on  the  supposition  that  filtration  effects  an  alteration  in  the  bouillon 
which  renders  it  unfit  for  the  growth  of  the  organism.  The  addi- 
tion of  an  equal  volume  of  horse  senim  to  a  bouillon  culture  of  B. 
cholersp  suis  did  not  facilitate  the  passage  of  the  organisms  through 
the  Pasteur-Chamberland  filter. 

Rabbits  were  injected  subcutaneously  with  10  c.  c.  of  filtered 
culture,  and  other  rabbits  were  injected  intravenously  and  intra- 
peritoneally  with  5  c.  c.  of  filtered  culture,  but  none  of  these  animals 
showed  any  ill  effects  from  the  injections.  Hogs  weighing  from  30  to 
40  pounds  were  injected  subcutaneously  with  20  c.  c.  of  filtered  cul- 
ture and  intravenously  with  10  c.  c.  of  filtered  culture,  but  were  not 


SUMMARY  AND   CONCLUSIONS.  31 

rendered  sick  thereby.  Collodion  sacs  containing  filtered  culture 
were  placed  in  the  abdominal  cavities  of  rabbits,  but  remained  sterile. 
Granules  were  noted  in  cultures  of  B.  cholerse  suis  and  in  the  filtrates 
from  these  cultures,  but  these  granules  did  not  develop  in  the  fil- 
trates nor  in  subcultures  made  from  these  filtrates.  These  granules 
were  also  shown  to  be  incapable  of  development  in  the  bodies  of  rabbits 
and  hogs. 

CONCLUSIONS. 

In  view  of  the  results  stated  above,  we  must  conclude — 

1.  That  Pasteur-Chamberland  filters  F  and  B  effectually  prevent 
the  passage  of  B.  cholerse  suis. 

2.  That  the  smaller  Berkefeld  laboratory  cylinders  vary  in  per- 
meability. 

3.  That  certain  of  the  Berkefeld  laboratory  cylinders  will  prevent 
the  passage  of  B.  cholerse  suis  when  a  limited  amount  of  material  is 
filtered. 

4.  That  the  granules  noted  in  cultures  of  B.  cholerse  suis  have  no 
significance  in  filtration  experiments  with  this  organism. 

5.  That  in  the  filtration  experiments  described  in  Bulletin  72  the 
filtrates  employed  did  not  contain  B.  cholerse  suis. 

6.  That  hog  cholera  is  due  to  an  ultra-visible  virus  sufficiently 
small  to  pass  through  the  pore:)  of  the  Chambsrland  filter. 

o 


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